A Case of Hairy Cell Leukemia Variant: Literature Analysis With Focus on Unmet Needs

Hairy cell leukemia variant (HCLv) is a sporadic, B-cell non-Hodgkin lymphoma classified under chronic lymphoproliferative disorders. HCLv usually presents with easy fatigue, dragging pain abdomen, anemia, splenomegaly, hepatomegaly, initially leukocytosis followed by leucopenia, hairy cells in the smear and bone marrow, and an increased risk of infections. There is hypercellular bone marrow, and cytopenias are secondary to hypersplenism. It is essential to differentiate HCL from disorders like classic hairy cell leukemia (HCLc), splenic marginal zone lymphoma, and splenic diffuse red pulp lymphoma, as these are biologically different, with divergent approaches and outcomes. HCLv is poorly responsive or primary refractory to standard purine analogs cladribine or pentostatin. It has lower response rates to even cladribine and rituximab combination, a standard of care for classic HCL with very good response rates. Here, we present a case of an elderly male who presented with splenomegaly and leukocytosis, diagnosed as HCLv, and was treated with a cladribine and rituximab-based regime but showed residual cells in bone marrow on flow cytometry at six months post-treatment. There were no residual cells in peripheral blood in flow cytometry. Various aspects of the disease are discussed here with a detailed literature analysis. There is a definite unmet need for research on better treatment options in HCLv to improve its overall outcome.


Introduction
Hairy cell leukemia (HCL) is a sporadic, indolent, B-cell non-Hodgkin lymphoma (NHL) that is classified under chronic lymphoproliferative disorders and comprises classical HCL (HCLc) and HCL-like disorders [1,2].Hairy cell leukemia variant (HCLv) is a provisional entity in the most recent revision of the WHO 2016 classification [1,2].It presents with anemia, thrombocytopenia, splenomegaly, hepatomegaly, and leukocytosis initially followed by leucopenia, increased risk of infections, and easy fatigue.It is essential to differentiate HCL from HCL-like disorders as these are biologically different, with divergent treatment approaches and prognoses.Morphologically, these disorders look similar, and immunophenotyping helps to distinguish between these entities.In 2008, the World Health Organization (WHO) reclassified the disorders into separate categories of lymphoproliferative disorders [3].The four immunophenotyping markers which help in differentiation are CD11C, CD103, CD25, and CD123.Each marker is assigned a score of 1 if expressed and 0 if not expressed.Classical HCL scores 3 to 4, whereas the HCL-like disorders have low immunological scores of 0 to 1 [1][2][3].HCLv is a mature lymphoid B-cell disorder characterized by the identification of hairy cells, a specific immunophenotypic profile, and a different clinical course than other disorders of a similar spectrum like HCLc, splenic marginal zone lymphoma (SMZL), and splenic diffuse red pulp lymphoma (SDRPL).HCLv patients do present with splenomegaly, leukocytosis, or infections.The circulating lymphoid cells have a morphology intermediate between prolymphocytes and hairy cells.The HCL immunological score is low (0 or 1), with no expression of CD25 and inconstant or weak CD123 expression [1][2][3].
HCLv is a more aggressive disease than HCLc, with a different clinical course and usually poorly responsive or primary refractory to standard treatment options.The response rate with purine analogs cladribine or pentostatin is greater than 90% for HCLc but in the 40-50% range for HCLv, with most responses being partial [4].In HCLv, the abnormal lymphoid cells do not usually express CD25, CD200, and CD123.The proto-oncogene B-Raf (BRAFV600E) gene mutation is not present in HCLv but is a hallmark of HCLc [1][2][3][4].Similarly, mitogen-activated protein kinase kinase 1(MAP2K1) gene mutations are present in about 30% of HCLv cases [1][2][3][4].HCLv is treated with more aggressive therapies with purine analogs and rituximab.The clinical course of HCLv is more aggressive, with a median overall survival (OS) of 9 years [1][2][3][4].Here, we present a case of HCLv who presented with splenomegaly and leukocytosis and was treated with a cladribine and rituximab-based regime.

Case Presentation
A 68-year-old male patient with Eastern Cooperative Oncology Group (ECOG) performance status of one presented with complaints of heaviness in the left upper side of the abdomen for one year, which was gradually progressive and worsened in the last four months.He also had B symptoms of fever and significant weight loss of more than 10% in the previous six months.His physical examination revealed massive splenomegaly, palpable 3 cm below the umbilicus.His hemogram showed a white blood cell (WBC) count at 25.8 x109/L, hemoglobin (Hb) level at 10.1 gm/dl, and platelet (PLT) count at 92 x 109/L.Differential counts showed an absolute neutrophil count of 1290 (N), 23994 lymphocytes (L), 516 monocytes (M), no eosinophils (E), and no basophils (B).Peripheral smear showed atypical lymphoid cells, round to oval nuclei with exemplary hairy processes, which are larger than the size of mature lymphocytes (Figure 1A).His bone marrow aspiration showed diluted marrow with suppression of erythroid and myeloid series, proliferation of lymphoid cells, mostly atypical lymphoid cells (68%), and absolute lymphocytosis.Lymphoid series accounted for 82% of all nucleated cells (Figure 1B).Bone marrow biopsy showed bony trabeculae with normocellular marrow, trilineage differentiation with predominantly normoblastic to micronormoblastic erythropoiesis, normal myelopoiesis, and adequate megakaryocytes.Focal marrow areas showed lymphoid cell clusters, intermediate size with coarse chromatin and perinuclear halo.There was no evidence of granulomatous inflammation (Figure 1C).Flow cytometry was done on bone marrow aspirate and showed negative for CD5, CD10, CD25, CD123, and CD200 and positive for CD103 and CD11c, suggesting the variant form of hairy cell leukemia.B-RAF mutation analysis by polymerase chain reaction (PCR) was negative.Contrast-enhanced computed tomography (CECT) of the neck, thorax, and abdomen revealed no significant abnormality or lymphadenopathy in the neck or thorax region.There was gross splenomegaly (21 cm) and hepatomegaly (19 cm).This patient received systemic therapy with one cycle of cladribine in a dose of 0.15 mg/Kg on days 1-5, with eight weekly doses of rituximab 375 mg/m2 beginning day one.The hemogram findings during treatment and at six months are shown in Table 1.Follow-up hemogram at six months showed a WBC count of 5.4 x109/L, a Hb level of 12.9 gm/dl, and a platelet count of 105 x 109/L.Differential counts showed 71% neutrophils (N), 17% lymphocytes (L), 9% monocytes (M), 03% eosinophils (E), and 00% basophils (B).Peripheral smear showed normocytic normochromic RBC (Figure 2A).Marrow aspiration findings showed cellular reactive marrow with mild erythroid hyperplasia, lymphoid series accounting for 12%, and no evidence of granulomas/residual malignancy (Figure 2B).Bone marrow biopsy findings showed bony trabeculae with normocellular marrow, trilineage differentiation with predominantly normoblastic to micronormoblastic erythropoiesis, normal myelopoiesis, and adequate megakaryocytes.Focal areas (1%) show lymphoid cell clusters, intermediate size with coarse chromatin, and perinuclear halo without any evidence of granulomatous inflammation (Figure 2C).CECT Abdomen shows hepatomegaly and splenomegaly (12.36cm) with standard shape, density, and enhancement.The splenomegaly had markedly reduced after treatment (Figures 3A, 3B).Flow cytometry revealed 0.5% B lymphoid cells on bone marrow aspirate with positive expression of hairy cell markers (CD103+CD11c+ CD20+ bright) (Figure 4).Flow cytometry identified 21% normal lymphocytes in peripheral blood consisting of 16% T cells, 4% NK cells, and 1% B cells.There was no evidence of any residual cells with hairy cell markers (Figure 5).The patient became asymptomatic at six months.Due to discrepant results for residual cells in the bone marrow and peripheral blood, he has been counseled for a second course of rituximab for eight doses to have undetected residual cells in the bone marrow.

Discussion
HCL-v, first described by Cawley et al. [5], occurs at an incidence of 0.03 per 100,000 persons per year, has a male predominance (ratio of 6:1), and constitutes around 0.4% of all chronic lymphoid malignancies [1][2][3][4][5].HCL-v is a disease of the elderly, with a median age of 71 [1][2][3][4].Initial symptoms are abdominal discomfort or pain, usually secondary to splenomegaly, hepatomegaly, and symptoms derived from cytopenias such as anemia, bleeding, and/or recurrent infections.Usually, diagnosis is delayed for many years due to nonspecific symptoms of weakness, easy fatigue, and dragging pain in the abdomen, which is attributed to advanced age.Sometimes, we find a history of blood transfusions because of anemia.The diagnosis warrants a detailed clinical history, physical examination, and investigations, including peripheral blood smear, bone marrow morphology, immunophenotyping, and molecular diagnostics.Peripheral blood morphology of HCLv typically shows small-to-medium lymphocytes with circumferential cytoplasmic projections.Patients usually present with massive splenomegaly, leukocytosis without monocytopenia, and a hypercellular bone marrow that can be easily aspirated.Patients develop cytopenias with time because of hypersplenism [1][2][3][4].
SMZL with villous lymphocytes is another indolent B-cell lymphoma that may be mistaken for HCL.However, there are unipolar or bipolar cytoplasmic projections rather than circumferential projections, as seen in HCL [1][2][3][4].SMZL is characterized by abnormal lymphoid cells with round nuclei, condensed chromatin, and basophilic cytoplasm with polar short villi (villous lymphocytes) in the peripheral blood [1][2][3][4].SDRPL is a provisional entity, very close if not identical to HCL-V.SDRPL could be the first step before the occurrence of HCLv and is characterized by the presence of a large proportion of small to medium-sized villous lymphoid cells in peripheral blood [1][2][3][4].The abnormal lymphoid cells have a polar distribution of the villi, and the nucleolus is small or invisible.The characteristics of HCLc, HCLv, SMZL, and SDRPL are further described in  HCLv is inherently aggressive and does not respond well to single-agent purine analogs.Cladribine given as a single five-day daily, the two-hour intravenous infusion has shown poorer response rates than HCLc [1][2][3][4].
The combination therapy of purine analogs with an anti-CD20 monoclonal antibody, rituximab, is the firstline treatment option for HCLv [6].In the largest phase 2 study by Chihara et al., 20 patients of HCLv received cladribine 0.15 mg/kg on days one to five with eight rituximab doses of 375 mg/m 2 at weekly intervals, beginning day one [6].Patients were eligible for a second rituximab course six months after cladribine if minimal residual disease (MRD) was detected in the blood.Lymphopenia and neutropenia are seen in 65-75% of patients by week two, which gradually improves by week four to six.The complete remission (CR) rate from the cladribine plus rituximab regime was 95%, and the median duration of CR was 70.1 months.The patients with TP53 mutations had a shorter progression-free survival (PFS) (median, 36.4 months vs. unreached; P = .0024)and OS (median, 52.4 months vs. unreached; P = .032).Similarly, subsets with MRDnegative CR at six months were showing a longer PFS (unreached vs. 17.4 months; P < .0001)and OS (unreached vs 38.2 months; P < .0001)[6].Very limited data is available regarding the median overall survival of patients in CR and patients without CR for other cases or case series since no follow-up treatment details are available.
Many treatment modalities have been attempted to treat HCLv with gradual evolution.Splenectomy is a potential treatment option shown in a study by Matutes et al. [7].It showed remission in 13 out of 19 patients lasting one to ten years, with a median of four years.Pentostatin and α-interferon, active in HCLc, have shown poor responses in HCLv [1][2][3][4]8].Allogenic marrow transplantation has been tried in a patient with HCLv, which achieved clinical remission for only 16 months [9].Splenic irradiation and alemtuzumab have also been attempted in HCLv with limited success [10,11].The bendamustine plus rituximab combination was attempted in three cases with a good response in first-line treatment in HCLv [12].Small studies showed the effect of the administration of cladribine and late rituximab after one month of cladribine [13].It is debatable whether starting rituximab concurrent with cladribine or after one month is preferable.There is a possibility of infections and fever with cladribine infusion, which makes concurrent rituximab administration difficult.
Due to the rarity of this disease and the limited number of studies, no clear second or subsequent lines of therapy are superior to retreatment with cladribine or any other choice.Once the patient relapses, several second-line options are rituximab, a monoclonal antibody against CD52 (alemtuzumab), and combination chemotherapies such as CHOP or CHOP-R [1][2][3][4]11].Newer therapies may include treatment with a recombinant immunotoxin with an anti-CD22 variable domain fused to a truncated Pseudomonas endotoxin, moxetumomab pasudotox, a Bruton's tyrosine kinase inhibitor, ibrutinib, and mitogen-activated protein kinase (MEK) inhibitors if MAP2K1 mutation is present [1][2][3][4].
The rearrangements expressing immunoglobulin variable heavy chain gene, VH4-34, commonly used in autoimmune disorders, were found in 40% of patients with HCLv versus 10% with HCLc in a study by Arons et al. [14].VH4-34+ patients were found to have higher white blood cell counts at diagnosis, a lower response rate and PFS, and shorter overall survival with first-line cladribine [14].
The cases of HCLv are scarce, leading to the publication of a limited number of case reports and small series.An extensive search for the concerned cases was done on the internet, and 28 case reports were found from 1996 to 2023, summarized in tabular form (Table 3) [3,4,[9][10][11][12].There are no phase 3 studies to date due to rare occurrences.Limited phase 2 studies and a series of cases are summarized in tabular form (Table 4) [6,8,13,[37][38][39][40][41][42].MRD measurements can be done with IHC, multicolor flow cytometry, or PCR methods.Our case showed a discrepancy in residual disease with 0.5% abnormal cells in bone marrow flow cytometry.However, no abnormal population of cells in flow cytometry was done on peripheral blood six months after therapy with cladribine and rituximab.Bone marrow examination is required to document remission post-treatment, and so it was performed, and flow was sent on the bone marrow sample.Flow cytometry or polymerase chain reaction on bone marrow is a better modality to detect MRD or early relapse in leukemias.We also wanted to check the residual disease status on blood as the largest study on HCLv by Chihara et al. had done flow on blood for remission status post-treatment [6].So, in our case, this discrepancy was picked up where bone marrow showed 0.5% residual cells while peripheral blood showed no residual cells.There is no consensus in the cases where the MRD discrepancy is noted.We have advised a second course of Rituximab to our case as bone marrow showed 0.5% residual disease and is presumed to be an early site of relapse even before the obvious signs of failure in peripheral blood.There are no standard comparisons for various methods of MRD assessment [43].There is no role of second rituximab in MRD-negative cases as of now, as MRD-negative patients were excluded from further doses of rituximab in the phase II study.Since HCLv differs from HCLc, we cannot use the same guidelines for both diseases.There are many dilemmas for a clinician which are yet to be answered.

Author
Which one is preferred out of concurrent and late rituximab with cladribine infusion?This also needs further research and consensus on whether to include rituximab maintenance in MRD-negative patients.Since it is an aggressive, poorly responsive disease, rituximab is a well-tolerated drug.Theoretically, there is no harm in adding rituximab maintenance in such cases to compensate for the poor disease biology.The role of adding Ibrutinib over second rituximab in MRD-positive cases at six months after cladribine and rituximab therapy needs further research and consensus.In MRD discrepant cases in bone marrow and blood, like in our case, what is ideal treatment is yet to be answered.There is no consensus on the preferred second-line therapy.To conclude, there are many unmet needs in the treatment part of HCLv, which warrants further research.

Conclusions
This case report summarizes the related scarce literature in one place and will likely help clinicians in decision-making and literature review for future cases.This case also highlights and replicates the poor response to the standard cladribine plus rituximab regime in such cases.This case reinforces the unmet need for future research in therapeutics for this aggressive disease.The role of MRD positivity, the timing of testing, the method of testing, and treatment implications are not standard at present, and this case reinforces the need for a clearer picture.

FIGURE 1 :
FIGURE 1: (A) Peripheral smear Leishman staining in 100X magnification showing atypical lymphoid cells with fine hairy processes (blue arrow); (B) bone marrow aspiration Leishman 400X magnification showing hairy cells having round to oval nucleus with moderate pale grey cytoplasm (blue arrow); (C) bone marrow biopsy hematoxylin and eosin (H&E) staining in 100X magnification showing cellular marrow, medium-sized lymphoid cells with oval nuclei, open chromatin, absent nucleoli, and a characteristic serrated cytoplasmic border

FIGURE 2 :FIGURE 3 :
FIGURE 2: (A) Peripheral smear Leishman staining in 10X magnification showing normal leukocyte count and distribution, (B) bone marrow aspiration Leishman 40X magnification showing normal hematopoietic cells and lymphocytes, and (C) bone marrow biopsy post-chemotherapy Leishman H&E 10X magnification showing remission of hairy cell leukemia